The biological role of the carboxyl-terminal extension of human chorionic gonadotropin [corrected] beta-subunit.
hCG is a member of a family of glycoprotein hormones which share a common alpha-subunit, but differ in their hormone-specific beta-subunits. The CG beta-subunit is unique in that it contains a hydrophilic carboxyl-terminal extension with four serine O-linked oligosaccharides. To examine the role of the O-linked oligosaccharides and the carboxyl-terminal extension of hCG beta on receptor binding, steroidogenesis in vitro, and ovulation induction in vivo, site-directed mutagenesis and gene transfer methods were used. Wild-type hCG alpha and hCG beta expression vectors were transfected into an O-glycosylation mutant Chinese hamster ovary cell line to produce intact dimer hCG lacking the beta-subunit O-linked oligosaccharide units. In addition, a mutant hCG beta gene (CG beta delta T) was generated which contained a premature termination signal at codon 115. This gene was cotransfected with the hCG alpha gene into Chinese hamster ovary cells to produce hCG dimer which lacked the carboxyl-terminal amino acids 115-145 of hCG beta (truncated hCG). The O-linked oligosaccharide deficient or truncated hCG derivatives were examined for their ability to bind to the mouse LH/hCG receptor and stimulate cAMP and steroidogenesis in vitro. These studies show that the O-linked oligosaccharides and carboxyl-terminal extension play a minor role in receptor binding and signal transduction. In contrast, comparison of the stimulatory effects of truncated and wild-type hCG in a rat ovulation assay in vivo via either intrabursal or iv injection revealed that the truncated derivative was approximately 3-fold less active than wild-type hCG. These findings indicate that the carboxyl-terminal extension of hCG beta and associated O-linked oligosaccharides are not important for receptor binding or in vitro signal transduction, but are critical for in vivo biological responses.
Matzuk
M M
MM
Department of Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110.
Hsueh
A J
AJ
Lapolt
P
P
Tsafriri
A
A
Keene
J L
JL
Boime
I
I
D000818:Animals; D002460:Cell Line; D055598:Chemical Phenomena; D002621:Chemistry; D006063:Chorionic Gonadotropin / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology*; D005260:Female; D005796:Genes; D006064:Gonadotropins, Equine / Q000494:pharmacology; D006801:Humans; D007016:Hypophysectomy; D007267:Injections; D007275:Injections, Intravenous; D009154:Mutation; D010053:Ovary / Q000187:drug effects; D010060:Ovulation / Q000187:drug effects; D011967:Receptors, Gonadotropin / Q000378:metabolism; D015398:Signal Transduction
D006063:Chorionic Gonadotropin; D006064:Gonadotropins, Equine; D011967:Receptors, Gonadotropin